bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Subunit redundancy within the NuRD complex ensures fidelity of ES cell lineage commitment Thomas Burgold1,4, Michael Barber1, Susan Kloet2, Julie Cramard1, Sarah Gharbi1, Robin Floyd1, Masaki Kinoshita1, Meryem Ralser1, Michiel Vermeulen2, Nicola Reynolds1, Sabine Dietmann1 and Brian Hendrich1, 3 1. Wellcome– MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR United Kingdom 2. Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University, 6525 GA Nijmegen, The Netherlands 3. Department of Biochemistry, University of Cambridge, Cambridge CB2 1QR United Kingdom 4. Present address: Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom Corresponding Author: Brian Hendrich, [email protected], +44 (0)1223 760205, @BDH_Lab Key Words: NuRD, Chromatin, ES Cell, Lineage Commitment, Transcription bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often present as multiple paralogous proteins in vertebrate complexes. In some cases these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within ES cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a complete NuRD null and are viable, allowing us to identify a previously undetected function for NuRD in maintaining differentiation trajectory during early stages of lineage commitment. 2 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Introduction Mammalian cells contain a number of proteins capable of using ATP hydrolysis to shift nucleosomes relative to the DNA sequence, thereby facilitating chromatin remodelling. In mammals, these ATP-dependent chromatin remodelling proteins usually exist within multiprotein complexes and play essential roles in the control of gene expression, DNA replication and repair (Hargreaves and Crabtree 2011; Narlikar et al. 2013; Hota and Bruneau 2016). NuRD (Nucleosome remodelling and deacetylation) is one such multiprotein complex which is unique in that it contains both chromatin remodelling and protein deacetylase activity. NuRD is highly conserved amongst metazoans and has been shown to play important roles in cell fate decisions in a wide array of systems (Denslow and Wade 2007; Signolet and Hendrich 2015). For example, in embryonic stem (ES) cells NuRD controls nucleosome positioning at regulatory sequences to finely tune gene expression (Reynolds et al. 2012; Bornelöv et al. 2018) and in somatic lineages NuRD activity has been shown to prevent inappropriate expression of lineage-specific genes to ensure fidelity of somatic lineage decisions (Denner and Rauchman 2013; Knock et al. 2015; Gomez-Del Arco et al. 2016; Loughran et al. 2017). It was recently demonstrated that this is achieved in both ES cells and B-cell progenitors by restricting access of transcription factors to regulatory sequences (Liang et al. 2017; Loughran et al. 2017; Bornelöv et al. 2018). Additionally, aberrations in expression levels of NuRD component proteins are increasingly being linked to cancer progression (Lai and Wade 2011; Mohd-Sarip et al. 2017). NuRD is comprised of two enzymatically and biochemically distinct subcomplexes: a chromatin remodelling and a deacetylase subcomplex. The chromatin remodelling subcomplex contains a nucleosome remodelling ATPase protein (Chd3/4/5) along with one 3 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. of the zinc finger proteins Gatad2a/b and the Doc1/Cdk2ap1 protein, while the deacetylase subcomplex contains class I histone deacetylase proteins Hdac1/2, the histone chaperones Rbbp4/7, the Metastasis Tumour Antigen family of proteins, Mta1, Mta2 and Mta3 and, in pluripotent cells, the zinc finger proteins Sall1/4 (Lauberth and Rauchman 2006; Allen et al. 2013; Kloet et al. 2015; Bode et al. 2016; Low et al. 2016; Miller et al. 2016; Spruijt et al. 2016; Zhang et al. 2016). These two subcomplexes are bridged by Mbd2/3, creating intact NuRD. While HDAC and RBBP proteins are also associated with other chromatin modifying complexes, the CHD, MBD, GATAD2 and MTA proteins are obligate NuRD components. Functional and genetic data indicate that the CHD4-containing remodelling subunit may be capable of functioning independently of intact NuRD (O'Shaughnessy and Hendrich 2013; O'Shaughnessy-Kirwan et al. 2015), though it is not clear whether the deacetylase subcomplex has any function outside of intact NuRD. Changes in subunit composition in large multiprotein, chromatin modifying complexes such as PRC1 and BAF has been shown to correlate with distinct changes in function to sites of action in the chromatin in a cell-type specific manner (Ho and Crabtree 2010; Morey et al. 2012). The NuRD complex might therefore be expected to show similar diversity in both composition and function and in fact, diversification of NuRD function has been described through differential incorporation of different isoforms of NuRD component proteins (Bowen et al. 2004). For example, Mbd2 and Mbd3 are mutually exclusive within NuRD (Le Guezennec et al. 2006). While Mbd2 is not required for mammalian development, Mbd3 is essential for early postimplantation mouse development (Hendrich et al. 2001). Mbd2/NuRD is a methyl-CpG binding co-repressor complex which is dispensable for early development but Mbd3/NuRD, a transcriptional modulator found at sites of active transcription, has been shown to play important roles in regulation of cell fate decisions in 4 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. multiple developmental systems (Feng and Zhang 2001; Reynolds et al. 2012; Gunther et al. 2013; Reynolds et al. 2013; Shimbo et al. 2013; Menafra et al. 2014). In brain development NuRD complexes containing either CHD3, CHD4 or CHD5 play distinct roles during cortical development (Nitarska et al. 2016). Further functional and biochemical diversification occurs through alternate use of the three MTA proteins within NuRD. MTA proteins function as a scaffold around which the deacetylase subcomplex is formed, comprising a 2:2:4 stoichiometry of MTAs:HDACs:RBBPs (Millard et al. 2013; Smits et al. 2013; Millard et al. 2016; Zhang et al. 2016). The three MTA proteins are highly conserved, differing from each other predominantly at their C-termini. The MTA1 protein was originally identified because of its elevated expression in metastatic cell lines (Toh et al. 1994), and subsequently all three MTA proteins have been shown to be up-regulated in a range of different cancer types (Covington and Fuqua 2014; Sen et al. 2014; Ma et al. 2016). The MTA1 and 3 proteins were shown to form distinct NuRD complexes in breast cancer cells and in B-cells and were recruited by different transcription factors to regulate gene expression (Mazumdar et al. 2001; Fujita et al. 2003; Fujita et al. 2004; Si et al. 2015). These studies did not detect biochemical interactions between MTA3 and the other MTA proteins, leading to the conclusion that MTA proteins are mutually exclusive within NuRD. In contrast, Mta1 was shown to interact with Mta2 in MEL cells, possibly indicating that mutual exclusivity may be cell type-specific (Hong et al. 2005). While all three Mta genes are expressed in ES cells, detailed biochemical analysis of interactions of MTA proteins with one another or with the various NuRD components in ES cells has not previously been described. Functional evidence does not support a strict lack of redundancy amongst MTA proteins during mammalian development. While zygotic deletion of Chd4 or Mbd3 results in 5 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. pre- or peri-implantation developmental failure respectively (Kaji et al. 2007; O'Shaughnessy-Kirwan et al. 2015), mice deficient in any one of the three MTA proteins show minimal phenotypes. Mice lacking either Mta1 or Mta3 are viable and fertile (Manavathi et al.